KR100879491B1 - Genetically engineered pyrroloquinoline quinone dependent glucose dehydrogenase comprising an amino acid insertion - Google Patents

Abstract

The present invention is Acinetobacter ( Acinetobacter) calcoaceticus ) improved variants of soluble pyrroloquinoline quinone (PQQ) -dependent glucose dehydrogenase (s-GDH) comprising amino acid insertions between positions 428 and 429 corresponding to known amino acid sequences, such s-GDH variants Gene encoding the protein, the protein of said s-GDH with improved substrate specificity for glucose, and different applications of such s-GDH variants, in particular for application to determine the concentration of sugars in the sample, inter alia, the concentration of glucose.

Description

Genetically engineered pyrroloquinoline quinone-dependent glucose dehydrogenases comprising amino acid insertions {GENETICALLY ENGINEERED PYRROLOQUINOLINE QUINONE DEPENDENT GLUCOSE DEHYDROGENASE COMPRISING AN AMINO ACID INSERTION}

The present invention is Acinetobacter ( Acinetobacter) calcoaceticus ) improved variants of soluble pyrroloquinoline quinone (PQQ) -dependent glucose dehydrogenase (s-GDH) comprising amino acid insertions between positions 428 and 429 corresponding to known amino acid sequences, such s-GDH variants Genes encoding, proteins of said s-GDH with improved substrate specificity for glucose, and different applications of such s-GDH variants, in particular for determining the concentration of sugars in the sample, inter alia, the concentration of glucose.

Measurement of blood glucose levels is of great importance for the clinical diagnosis and management of diabetes. Around 150 million people worldwide have diabetes, a chronic disease, and the number could double by 2025, according to the WHO. Although diabetes is easily diagnosed and treated, successful long-term management requires low-cost diagnostic tools that can record blood glucose levels quickly and accurately. PQQ-dependent glucose dehydrogenase (EC 1.1.99.17) catalyzes the reaction in which glucose is oxidized to gluconolactone. Thus, this type of enzyme is used to measure blood glucose. One such tool is soluble glucose dehydrogenase (s-GlucDOR, EC 1.1.99.17), a pyrroloquinoline quinone-containing enzyme originally derived from acinetovector chalcocaceticus.

Quinoproteins use quinones as cofactors to oxidize alcohols, amines and aldoses to their corresponding lactones, aldehydes and aldol acids (Duine, JA Energy generation and the glucose dehydrogenase pathway in Acinetobacter in "The Biology of Acinetobacter" ( 1991) 295-312, New York, Plenum Press; Duine, JA, Eur J Biochem 200 (1991) 271-284; Davidson, VL, in "Principles and applications of quinoproteins" (1993) the whole book, New York, Marcel Dekker; Anthony, C, Biochem. J. 320 (1996) 697-711; Anthony, C. and Ghosh, M., Current Science 72 (1997) 716-727; Anthony, C, Biochem. Soc. Trans. 26 ( 1998) 413-417; Anthony, C. and Ghosh, M., Prog.Biophys.MoI. Biol. 69 (1998) 1-21). Among the quinoproteins, the largest subgroup contains noncovalently bound 2,7,9-tricarboxy-lH-pyrrolo [2,3-f] quinoline-4,5-dione (PQQ) cofactors. (Duine 1991, supra). All bacterial quinone glucose dehydrogenases known to date belong to this subgroup with PQQ as a cofactor (see Anthony and Ghosh 1997, Goodwin, PM and Anthony, C, Adv. Microbiol. Physiol. 40 (1998) 1 -80; Anthony, C, Adv. In Phot, and Resp. 15 (2004) 203-225).

Two kinds of PQQ-dependent glucose dehydrogenase (EC 1.1.99.17) have been characterized in bacteria: one is membrane binding (m-GDH) and the other is soluble (s-GDH). The two types did not share any significant sequence homology (Cleton-Jansen, AM, et al., Mol. Gen. Genet. 217 (1989) 430-436; Cleton-Jansen, AM, et al., Antonie Van Leeuwenhoek 56 (1989) 73-79; Oubrie, A., et al., Proc. Natl. Acad. Sci. USA 96 (1999) 11787-11791). They differ in both their kinetic and immunological properties (Matsushita, K., et al., Bioscience Biotechnol. & Biochem. 59 (1995) 1548-1555). m-GDHs are common in gram-negative bacteria, while s-GDH is A. calcicoaceticus (Duine, JA, 1991a; Cleton-Jansen, AM et al., J. Bacteriol. 170 (1988) 2121-2125; Matsushita and was only found in Adachi, 1993) and A. Bauer maniyi (A. baumannii) (JP 11243949) and periplasmic space of ahsine Saturday vectors such strains.

Through the sequence database, two sequences homologous to the full-length A. Calcoaceticus s-GDH were identified in E. coli K-12 and Cynecosistis species. Additionally, A. Sethi knife core coarse s-GDH and two incomplete sequences homologous to the P. Additionally Ke Rouge labor (P. aeruginosa) and Bode telra pertussis (Bordetella pertussis ) (Oubrie et al. 1999 a, b, c) and Enterobacter intermedium (Kim, CH. et al., Current Microbiol. 47 (2003) 457-461), respectively. The deduced amino acid sequences of these four uncharacterized proteins are closely related to A. Calcoaceticus s-GDH and many residues within the putative active site are completely conserved. Such homologous proteins have similar structures and can promote similar PQQ-dependent responses (Oubrie et al., 1999 a, b, c; Oubrie A., Biochim. Biophys. Acta 1647 (2003) 143-151; Reddy, S., and Bruice, TC, J. Am. Chem. Soc. 126 (2004) 2431-2438; Yamada, M. et al., Biochim. Biophys. Acta 1647 (2003) 185-192).

The bacterial s-GDH and m-GDH have been found to have quite different sequences and different substrate specificities. For example, A. Calcoaceticus contains two different PQQ-dependent glucose dehydrogenases, one is m-GDH active in vivo, and the other is s- which can only display in vitro activity. It is called GDH. Cleton-Jansen et al., 1988; 1989 a, b cloned a gene encoding two GDH enzymes and determined the DNA sequence of both of these GDH genes. There is no apparent homology between m-GDH and s-GDH and it is confirmed that m-GDH and s-GDH represent two completely different molecules (Laurinavicius, V., et al, Biologija (2003) 31-34).

The s-GDH gene from A. Calcoaceticus was cloned into E. coli. After being produced intracellularly, s-GDH travels through the cytoplasmic membrane to the periplasm (Duine, JA, Energy generation and the glucose dehydrogenase pathway in Acinetobacter in "The Biology of Acinetobacter" (1991) 295-312, New York , Plenum Press; Matsushita, K. and Adachi, O., Bacterial quinoproteins glucose dehydrogenase and alcohol dehydrogenase in "Principles and applications of Quinoproteins" (1993) 47-63, New York, Marcel Dekker). Like native s-GDH from A. chalcoaceticus, recombinant s-GDH expressed in E. coli is a homodimer with three calcium ions and one PQQ molecule per monomer (Dokter, P. et al. , Biochem. J. 239 (1986) 163-167; Dokter, P. et al., FEMS Microbiol. Lett. 43 (1987) 195-200; Dokter, P. et al., Biochem. J. 254 (1988) 131-138; Olsthoorn, A. and Duine, JA, Arch.Biochem.Biophys. 336 (1996) 42-48; Oubrie, A., et al., J. MoI. Biol. 289 (1999) 319-333, Oubrie, A., et al., Proc. Natl. Acad. Sci. USA 96 (1999) 11787-11791, Oubrie, A., et al., Embo J. 18 (1999) 5187-5194). s-GDH oxidizes a wide range of monosaccharides and disaccharides to the corresponding ketones, which are further hydrolyzed to aldonic acid, which is also PMS (phenazine methosulfate), DCPIP (2,6-dichlorophenolrindophenol ), WB (Wurster's blue) and short-chain ubiquinones such as ubiquinone Q1 and ubiquinone Q2 (Matsushita, K., et al., Biochem. 28 (1989) 6276-6280; Matsushita, K., et al., Antonie Van Leeuwenhoek 56 (1989) 63-72), several artificial electron acceptors such as N-methylphenazonium methyl sulphate (Olsthoorn, AJ and Duine, JA, Arch. Biochem. Biophys. 336 (1996) 42-48; Olsthoorn, AJ and Duine, JA, Biochem. 37 (1998) 13854-13861) and conductive polymers (Ye, L., et al., Anal. Chem. 65 (1993) 238). In view of the high specific activity of s-GDH against glucose (Olsthoorn, AJ and Duine, JA, (1996), see above) and its broad artificial electron acceptor specificity, this enzyme is useful for analytical applications, in particular diagnostics. Suitable for use in test strips or (bio-) sensors for measuring glucose in applications (Kaufmann, N. et al., Development and evaluation of a new system for determining glucose from fresh capillary blood and heparinised blood in "Glucotrend" (1997) 1-16, Boehringer Mannheim GmbH; Malinauskas, A .; et al., Sensors and Actuators, B: Chemical BlOO (2004) 395-402).

Glucose oxidation involves at least three significantly different groups of enzymes: NAD / P-dependent glucose dehydrogenase, flavoprotein glucose oxidase or quinoprotein GDH (Duine, JA, Biosens. Bioelectronics 10 (1995) 17-23 Can be catalyzed by Somewhat slow self-oxidation of the reduced s-GDH was observed, indicating that oxygen is a very poor receptor for s-GDH (Olsthoorn and Duine, 1996). s-GDH can efficiently provide electrons from reduced quinones to mediators such as PMS, DCPIP, WB and short-chain ubiquinones such as Q1 and Q2, but this cannot efficiently provide electrons directly to oxygen.

For example, conventional test strips and sensors for monitoring glucose levels in blood, serum and urine from diabetic patients use glucose oxidase. The performance of the enzyme depends on the oxygen concentration. Glucose measurements at different altitudes with different oxygen concentrations in air can lead to erroneous results. The main advantage of PQQ-dependent glucose dehydrogenase is its independence from oxygen. This important feature is described, for example, in US Pat. No. 6,103,509, where several features of membrane bound GDH are investigated.

An important contribution to this area was the use of s-GDH with appropriate mediators. s-GDH based test strip devices and analysis methods are described in detail in US Pat. No. 5,484,708. This patent also includes the setup of an assay for glucose measurement and the manufacture of s-GDH-based test strips. The methods described therein and the literature cited therein are incorporated by reference.

Other patents or applications related to this field, including specific data on various methods of application for enzymes with glucose dehydrogenase activity, are described in US 5,997,817; US 6,057,120; EP 0 620 283; And JP 11-243949-A.

A commercially available system using s-GDH and an indicator (see Kaufmann et al. 1997 above) that produces a color change when the reaction occurs is Glucotrend ^® distributed by Roche Diagnostics GmbH.

In addition to the advantages described above for the use of PQQ dependent s-GDH, the disadvantages in measuring glucose should also be considered. The enzyme has a rather broad substrate range compared to m-GDH. That is, s-GDH oxidizes not only glucose but also some other sugars, including maltose, galactose, lactose, mannose, xylose and ribose (Dokter et al. 1986 a; Oubrie A., Biochim. Biophys. Acta 1647 (2003) 143-151). Reactivity to sugars other than glucose can in some cases reduce the accuracy of measuring blood glucose levels. In particular, a patient in peritoneal dialysis treated with Icodextrin (glucose polymer) may contain high levels of other sugars, especially maltose, in their body fluids, for example blood (Wens, R., et al., Perit.Dial) Int. 18 (1998) .603-609).

Thus, clinical samples, such as obtained from diabetic patients, in particular patients with renal complications and especially patients during dialysis, may contain significant levels of other sugars, in particular maltose. Glucose measurements in samples obtained from these critical patients can be compromised by maltose (Davies, D., Perit. Dial. Int. 14 (1994) 45-50; Frampton, JE; and Plosker, G. L, Drugs 63 (2003) 2079-2105).

There are very few reports in the literature on attempts to produce modified PQQ-dependent s-GDH with altered substrate specificity. Igarashi, S., et al., Biochem. Biophys. Res. Commun. 264 (1999) 820-824 report that point mutation introduction at the Glu277 position results in mutations with altered substrate specificity profiles.

Sode (EP 1 176 202) reports that certain amino acid substitutions in s-GDH result in s-GDH mutations with improved glucose affinity. In EP 1 167 519 the same author reports on mutant s-GDH with improved stability. In addition, the same author reports on other s-GDHs with improved affinity for glucose in JP2004173538.

Kratzsch, P. et al. (WO 02/34919) found that the specificity of s-GDH to glucose can be improved by amino acid substitution at certain positions of s-GDH compared to other sugar substrates, especially compared to maltose. Report it.

Takeshima, S. et al. (EP 1 367 120) report on mutant s-GDH comprising amino acid insertions or specific amino acid substitutions between positions 427 and 428, corresponding to known amino acid sequences from acinetovector chalcocetitices. .

However, although quite a few improvements have been reported for the generation of mutations or variants of s-GDH with improved properties, further alternatives and / or further improvements are still needed.

There are many demands and clinical needs for additional mutations or variant forms of s-GDH that result in improved specificity for glucose as a substrate, alone or in combination with known mutations.

An object of the present invention is a novel mutation or variant of s-GDH, alone or in combination with known mutations, resulting in substrate specificity for glucose which is significantly improved compared to other selected sugar molecules such as galactose or maltose. To provide.

Surprisingly, amino acid insertions between positions 428 and 429 of s-GDH, which correspond to the amino acid sequences known from Acinetovector chalcocaceticus, can significantly improve the substrate specificity of s-GDH for glucose over other sugars. Thus, it has been found possible to at least partially solve the above-mentioned problems known in the art.

Substrate specificity for glucose compared to other selected sugar molecules is markedly improved by providing an insertion modification of s-GDH according to the present invention, as described in the following and appended claims.

Because of the improved substrate specificity of the new form of s-GDH, significant technological advances are possible for glucose measurement in a variety of applications. The improved s-GDH variant can be very advantageously used for the specific detection or measurement of glucose in biological samples, especially in test strip devices or biosensors.

Summary of the invention:

The present invention relates to a variant of the soluble form of EC 1.1.99.17, also known as PQQ-dependent soluble glucose dehydrogenase (s-GDH), wherein the variant is an s-GDH wild type sequence (SEQ) from A. Calcoaceticus. One or more amino acid residue insertions between amino acid positions corresponding to positions 428 and 429 of ID NO: 2), and optionally one or more amino acid substitutions, preferably substitutions at positions 328 and 428.

Also provided are preferred variants of s-GDH that exhibit improved properties, particularly increased specificity for glucose, and polynucleotide sequences encoding such variants, expression vectors comprising such polynucleotide sequences, and host cells comprising the expression vectors. do.

The invention further relates to the use of the variant according to the invention in a method of measuring glucose, in particular by a test strip device or a biosensor.

The following examples, references, sequence listings and figures are provided to aid the understanding of the present invention, the exact scope of which is set forth in the appended claims. Changes may be made in the methods described without departing from the spirit of the invention.

Figure 1: Protein sequences of A. chalcoaceticus PQQ-dependent s-GDH (top) and A. Baumaniyi s-GDH (bottom) aligned according to sequence homology.

2: Illustration of pACSGDH referred to in Example 1, each comprising a wild type or mutant DNA sequence of soluble PQQ-dependent glucose dehydrogenase.

3: Nucleotide (DNA) sequence of the pACSGDH vector referred to in Example 1, comprising the wild type DNA sequence of soluble PQQ-dependent glucose dehydrogenase.

Detailed description of the invention:

As mentioned above, two completely different quinoprotein enzyme types (membrane binding and soluble) with glucose dehydrogenase activity were classified together under EC 1.1.99.17. These two types do not look related to each other.

Only GDH (s-GDH) in soluble form is suitable for the purposes of the present invention, and improved variants thereof are described below.

In a first embodiment, the present invention relates to a variant of the soluble form of EC 1.1.99.17, also known as PQQ-dependent soluble glucose dehydrogenase (s-GDH), wherein the variant is A. Calcoaceticus (SEQ ID). NO: 2) one or more amino acid residue insertions between amino acid positions corresponding to positions 428 and 429 of the s-GDH wild type sequence known from 2) and optionally further comprising a modification comprising one or more amino acid substitutions.

Preferably, only one amino acid is inserted between amino acid positions corresponding to positions 428 and 429 of the s-GDH wild type sequence known from A. Calcoaceticus (SEQ ID NO: 2).

The s-GDH variant comprising an amino acid insertion between amino acid positions corresponding to positions 428 and 429 of the s-GDH wild type sequence known from A. Calcoaceticus (SEQ ID NO: 2), wherein the inserted amino acid is leucine, More preferably, it is selected from the group consisting of phenylalanine, methionine and proline. Preferably the inserted amino acid is proline.

It is known in the art that wild-type DNA-sequences of soluble PQQ-dependent glucose dehydrogenase can be isolated from strains of acinetovectors. Most preferred is the isolation from acinetovector chalcoceticus-type strain LMD 79.41. The sequence of wild type s-GDH (mature protein) is provided in SEQ ID NO: 2. Other LMD strains of acinetovector can also be used as a source of wild type s-GDH. Such sequences can be aligned to sequences obtained from A. Calcoaceticus for sequence comparison. For example, the DNA-library of other bacterial strains can be screened, as described for E. coli K12 (Oubrie, A., et al., J. MoI. Biol. 289 (1999) 319-333), It is also possible to identify sequences related to s-GDH in such genomes. Such sequences and homologous sequences not yet identified can be used to generate s-GDH with improved substrate specificity.

In the sense of the present invention the term “variant” is used between amino acid positions corresponding to positions 428 and 429 of s-GDH known from A. Calcoaceticus (SEQ ID NO: 2), as compared to the corresponding wild type sequence. It relates to proteins with amino acid insertions.

"Mutations" in the sense of the present invention are directed to s-GDH having one or more amino acid substitutions relative to the corresponding wild-type sequence as compared to the s-GDH wild-type sequence known from A. Calcoaceticus (SEQ ID NO: 2). Related.

The term “variant” or “mutant” is used between amino acid positions corresponding to positions 428 and 429 of the s-GDH wild type sequence known from A. Calcoaceticus (SEQ ID NO: 2), as compared to the corresponding wild type sequence. All commonly apply to s-GDH proteins with amino acid insertions and with one or more amino acid substitutions.

In a further preferred embodiment, the enzymatic or functional properties of the improved modification of s-GDH are compared with wild type enzymes or mutations without amino acid insertions between positions 428 and 429, respectively.

Preferred variants according to the invention are characterized by having at least two times improved substrate specificity for glucose compared to one or more selected other sugar substrates, relative to the corresponding wild type enzyme.

To calculate substrate specificity or cross-reactivity, one easy way is to set glucose measured as substrate to 100% activity and compare the activity measured with other selected sugars to glucose values. To avoid occasional verbosity, one does not refer to glucose on the one hand, or to other sugar substrates selected on the other, and simply uses the term specificity.

Those skilled in the art will understand that enzymatic activity is best compared at equimolar concentrations of substrate molecules investigated using clearly defined assay conditions. Otherwise, a correction for the concentration difference should be made.

Standardized and clearly defined assay conditions should be selected to assess substrate specificity (improvement). The enzymatic activity of s-GDH against glucose as a substrate and other selected sugar substrates are measured as described in Example 7.

Based on the measurement of enzymatic activity against glucose or other sugars selected, preferably maltose, cross-reactivity (and improvement thereof) was evaluated.

The s-GDH (cross) reactivity for the selected sugars was calculated as follows:

Cross-reactivity [%] = (selected sugar activity / glucose activity) x 100%.

The (cross) reactivity of maltose of wild type s-GDH according to the above formula was measured to be about 105%. Wild-type s-GDH (cross) reactivity against galactose was determined to be about 50% (see Table 1).

The (improved) specificity was calculated according to the formula:

Compared with wild-type enzymes, the s-GDH form with a 10-fold or more improvement in specificity for glucose (maltose / glucose) over maltose, thus maltose-based substrates, has a 10-fold activity of glucose as a substrate. , 5% or less. Or, for example, if the mutant s-GDH has cross-reactivity to maltose of 20% (measured and calculated as described above), then this mutation is 5.25 times improved substrate compared to wild type s-GDH Specificity (maltose / glucose).

The term "specific activity" for a substrate is well known in the art and it is preferably used to describe the enzymatic activity per amount of protein. Various methods for measuring the specific activity of GDH molecules using glucose or other sugars as substrates (see above, Igarashi, S., et al., (1999)) are used in the art. One of the methods available for such a measurement is described in detail in the Examples section.

While it is possible to select a number of different sugar molecules and investigate the glucose specificity of s-GDH compared to any such sugar molecule selected, it is desirable to select a clinically appropriate sugar molecule for such a comparison. Preferred selection sugars are selected from the group consisting of mannose, allose, galactose, xylose, and maltose. Preferably maltose or galactose is selected and the mutant s-GDH is tested for improved substrate specificity for glucose as compared to galactose or maltose. In a further preferred embodiment the sugar selected is maltose.

For example, for maltose versus glucose, the improvement in glucose specificity of the s-GDH variant according to the invention has been found to be quite significant. Thus, it is more desirable that the substrate specificity for glucose is improved by at least threefold as compared to the substrate specificity for one or more of one or more selected other sugar substrates. Other preferred embodiments include s-GDH mutations characterized by improved substrate specificity for glucose, at least 5 times higher, or preferably at least 10 times higher than other sugar molecules selected.

Mutations in s-GDH result in many cases of enzyme variants with dramatically reduced specific activity against glucose substrates. However, a more than 10-fold reduction (absolute or comprehensive) of specific activity on glucose substrates may be important for routine applications. Thus, it is preferred that s-GDH with improved specificity for glucose substrates exhibits at least 10% specific activity against glucose measured with wild type enzymes. It is more preferred that such mutant enzymes exhibit at least 20% or more preferably at least 30% of each glucose activity of wild type s-GDH.

More preferably, maltose specific activity is no more than 10% or only 5% or less of the maltose specific activity measured for the corresponding wild type enzyme on a test strip or liquid experiment, and the specific activity for glucose is glucose of the corresponding wild type enzyme. Mutations ≥ 10% compared to the specific activity for.

Substrate specificity of the s-GDH variant comprising an insertion between positions 428 and 429 may be further improved by further modifying this variant to additionally include one or more amino acid substitutions at the specifically defined specific amino acid position. Found.

Amino acid positions known from the wild-type sequence SEQ ID NO: 2 of s-GDH isolated from the Acinetovector chacoaceticus type strain LMD 79.41 are mentioned and the achievement of the present invention is described in greater detail. Amino acid positions in different s-GDH isolates corresponding to the positions of SEQ ID NO: 2 are readily identified by appropriate sequence comparison.

Multiple alignment and comparison of the s-GDH sequence with the wild type sequence of SEQ ID NO: 2 was performed with the PileUp program of GCG Package Version 10.2 (Genetics Computer Group, Inc.). PileUp is described by Feng, D. F. and Doolittle, R. F., J. MoL Evol. 25 (1987) 351-360, using the simplification of the progressive alignment method to generate multiple sequence alignments, whereby measurement matrices for the same, similar or different amino acid residues are defined accordingly. The process begins with two pair alignments of the two most similar sequences, resulting in a population of two aligned sequences. This population may be aligned to the next most relevant sequence or to a population of aligned sequences. Two populations of sequences can be aligned by simple extension of two pair alignments of two separate sequences. Final alignment is achieved by a series of progressive two-pair alignments that include an incrementally dissimilar sequence and population until all sequences are included in the final two-pair alignment. In this way, it can be readily identified that the positions within other homologous s-GDH molecules correspond to the positions provided for A. chalcoacetis in SEQ ID NOs: 1 and 2, respectively. Thus, the amino acid position provided herein may be understood as the amino acid position of SEQ ID NO: 2 or a corresponding position in other homologous s-GDH molecules.

Mutations comprising s-GDH comprising amino acid substitutions at positions corresponding to position 348 and amino acid insertions between positions 428 and 429 of s-GDH have a markedly positive effect with respect to specificity for glucose Found. As shown in Table 1, several s-GDH variants with improved specificity for glucose were identified and generated. The improvement in glucose specificity for variant s-GDH appears as long as the amino acid at threonine position 348 is substituted with an appropriate other amino acid and the appropriate amino acid is inserted between positions 428 and 429. Thus a very preferred embodiment of the invention inserts between amino acids 428 and 429 of s-GDH known from A. Calcoaceticus (SEQ ID NO: 2) and additionally replaces amino acid residue substitutions at amino acid positions corresponding to positions 348 It relates to a variant protein of PQQ-dependent s-GDH comprising.

S-GDH further substitution at the amino acid position corresponding to positions 169, 171, 245, 341, 349 and / or 428 of SEQ ID NO: 2 includes an insertion between amino acids 428 and 429 and an amino acid residue substitution at position 348 It has been found advantageous in an effort to further improve the specificity of the variant to glucose.

It is known in the art that none of the insertions of amino acids between positions 428 and 429 of s-GDH isolated from the acinetovector chacoaceticus type strain LMD 79.41 or the threonine residues at position 348 contribute to substrate binding of s-GDH. Known (Oubrie, A., et al, Embo J. 18 (1999) 5187-5194; Oubrie, A. and Dijkstra, BW, Protein Sci. 9 (2000) 1265-1273). There is no chemical or physical explanation why the two variants of s-GDH improve substrate specificity for glucose compared to other sugar molecules of interest, especially compared to maltose.

In a further preferred embodiment the s-GDH variant is characterized in that the threonine amino acid residue at position 348 is replaced with an amino acid residue selected from the group consisting of alanine, glycine and serine. In a more preferred embodiment, threonine is substituted at position 348 using glycine. The term T348G is known to those skilled in the art and refers to the replacement of threonine with glycine at the 348 position.

A further preferred embodiment is a variant of the soluble form of EC 1.1.99.17, also known as PQQ-dependent soluble glucose dehydrogenase (s-GDH), which variant is from A. Calcoaceticus (SEQ ID NO: 2). One or more amino acid residue insertions between amino acid positions corresponding to positions 428 and 429 of the known s-GDH wild type sequence and one or more amino acid residue substitutions at amino acid positions corresponding to position 428. Preferably, the substitution of asparagine at position 428 is by leucine, proline and valine. More preferred substitution at position 428 is with proline.

One group of preferred s-GDH variants according to the present invention include substitution of amino acid residues at position 348, and / or amino acid substitutions at position 428 and amino acid insertions between positions 428 and 429. Such variants optionally comprise one or more amino acid substitutions at amino acid positions corresponding to positions 169, 171, 245, 341, and / or 349 of the s-GDH wild type sequence known from A. Calcoaceticus (SEQ ID NO: 2). It may be further modified to include.

When the amino acid corresponding to position 169 of the s-GDH wild type sequence known from A. chalcoceticus (SEQ ID NO: 2) is substituted for a variant of the invention, the naturally occurring amino acid leucine is substituted with phenylalanine, tyrosine or tryptophan It is preferable. Preferred substitution at position 169 is with phenylalanine.

When the amino acid corresponding to position 171 of the s-GDH wild-type sequence known from A. chalcoceticus (SEQ ID NO: 2) is substituted for a variant of the present invention, the group of naturally occurring amino acids tyrosine consists of alanine, methionine, glycine It is preferably substituted by an amino acid selected from the group consisting of. More preferred substitutions at position 171 are with glycine.

When the amino acid corresponding to position 245 of the s-GDH wild type sequence known from A. chalcoceticus (SEQ ID NO: 2) is substituted for a variant of the invention, the naturally occurring amino acid glutamic acid is substituted with aspartic acid, asparagine or glutamine It is desirable to be. More preferred substitution at position 245 is with aspartic acid.

When the amino acid corresponding to position 341 of the s-GDH wild type sequence known from A. chalcoceticus (SEQ ID NO: 2) is substituted for a variant of the invention, the naturally occurring amino acid methionine is substituted with valine, alanine, leucine or isoleucine. It is preferable to substitute by. More preferred substitution at position 341 is with valine.

When the amino acid corresponding to position 349 of the s-GDH sequence corresponding to the known s-GDH wild type sequence from A. Calcoaceticus (SEQ ID NO: 2) is substituted for a variant of the invention, the naturally occurring amino acid valine is alanine It is preferable to substitute by glycine. More preferred substitutions at the 349 position are by alanine.

As described in WO 02/34919, amino acid substitution at position 348 of the s-GDH sequence corresponding to the wild-type sequence isolated from A. Chalcoaceticus can be used to significantly improve glucose specificity of s-GDH. have. Those skilled in the art will find in WO 02/34919 other suitable positions which may be combined and substituted with the insertion according to the invention.

In a further preferred embodiment, the s-GDH variant according to the invention is in addition to the insertion between 428 and 429 amino residues, at the amino acid position of the s-GDH wild type sequence known from A. Calcoaceticus (SEQ ID NO: 2). At least two amino acid substitutions selected from the group consisting of the corresponding 171, 245, 341, 348 and 349 positions.

In another further preferred embodiment, the s-GDH variant according to the present invention, in addition to the insertion between 428 and 429 amino residues, comprises a s-GDH wild type sequence known from A. Calcoaceticus (SEQ ID NO: 2). At least three amino acid substitutions selected from the group consisting of positions 171, 245, 341, 348, and 349 corresponding to amino acid positions.

As will be appreciated by those skilled in the art, it is possible to take amino acid substitutions, for example silent mutations, which do not significantly affect the properties of s-GDH. However, variants according to the invention have up to 45 amino acid exchanges as compared to SEQ ID NO: 2. Preferably the variant will comprise no more than 20 amino acid substitutions, more preferably no more than 10 amino acid substitutions.

S-GDH variants according to the invention are provided in the Examples section. These variants also represent preferred embodiments of the invention. Variants with the least glucose interference found to date include insertions between amino acids 428 and 429, preferably by proline and Yl71G, E245D, M341V and T348G mutations or L169F, Y171G, E245D, M341V and T348G mutations, respectively. These two variants are further preferred embodiments of the present invention.

Amino acid sequencing is a key relevant position where the sequence motifs found in wild type s-GDH from A. chalcoceticus on the one hand and A. Baumaniyi on the one hand improve the specificity for glucose as identified herein. That is, it appears to be very conservative around the insertion site around positions 428 and 429 corresponding to wild type s-GDH from A. Calcoaceticus.

Variants of PQQ-dependent s-GDH comprising the amino acid sequence of AGNXaaVQK (SEQ ID NO: 2) represent preferred embodiments of the present invention. SEQ ID NO: 2 corresponds to positions 426-431 of A. chacoaceticus wild type s-GDH or positions 427-432 of A. Baumaniyi wild type s-GDH, but positions 428 and 429 (A. chacoaceticus) Each insert of one amino acid (Xaa) between or between positions 429 and 430 (A. Baumaniyi).

In a preferred embodiment, the invention relates to an s-GDH variant comprising the G-N-Xaa-V-Q-K-D (SEQ ID NO: 11) sequence. Preferably, the s-GDH variant containing SEQ ID NO: 11 is characterized in that the inserted amino acid Xaa is selected from the group consisting of leucine, proline, phenylalanine and methionine, more preferably Xaa is a proline residue.

As mentioned above, the amino acid asparagine at position 428 of the wild type s-GDH may be amino acid substituted. In this case SEQ ID NO: 11 will include substituted amino acids instead of P428.

Several possibilities for producing mutant proteins are known in the art. Based on the important findings of the present invention that initiate amino acid insertions between positions 428 and 429, which are critically important, those skilled in the art can now easily produce more suitable variants of s-GDH. Such variants are for example random mutagenesis (Leung, DW, et al., Technique 1 (1989) 11-15) and / or directed mutagenesis (Hill, DE, et al., Methods Enzymol. 155 (1987) 558 -568). Alternative methods of preparing proteins with the desired properties are to provide chimeric constructs comprising sequence elements from two or more different sources, or to fully synthesize the appropriate s-GDH gene. Such methods known in the art are used in conjunction with the information disclosed herein, eg, mutations in s-GDH comprising additional amino acid substitutions with the disclosed insertion between positions 428 and 429 of SEQ ID NO: 2 Or provide variants.

S-GDH variants according to the invention can be generated starting with the s-GDH gene isolated from, for example, the acetovector chalcocetiticus-type strain LMD 79.41, and starting from homologous sequences. "Homologous" in the context of the present application means to include an s-GDH amino acid sequence having at least 90% identity as compared to SEQ ID NO: 2. In other words, after proper alignment using the PileUp program, at least 90% of the amino acids of such homologous s-GDH are identical to the amino acids set forth in SEQ ID NO: 2.

It will be appreciated that variations in DNA and amino acid sequences may exist naturally or may be intentionally introduced using methods known in the art. Such mutations can result in amino acid differences of up to 10% of the total sequence, as compared to SEQ ID NO: 2, due to the deletion, substitution, insertion, inversion or addition of one or more amino acid residues in the sequence. Such amino acid substitutions can be carried out, for example, on the basis of the similarity of the polar, charge, solubility, hydrophobicity, hydrophilicity and / or amphoteric properties of the involved residues. For example, negatively charged amino acids include aspartic acid and glutamic acid; Positively charged amino acids include lysine and arginine; Amino acids having uncharged polar head groups or nonpolar head groups with similar hydrophilicity values include: leucine, isoleucine, valine, glycine, alanine, asparagine, glutamine, serine, threonine, phenylalanine, tyrosine. Other contemplated variations include salts and esters of the polypeptides described above, and precursors of the polypeptides described above, for example precursors with N-terminal substitutions such as methionine, N-formylmethionine, used as leading sequences. Such variations may be made without departing from the scope and spirit of the invention.

According to methods known in the art according to the methods provided in the Examples section, it is possible to obtain polynucleotide sequences encoding any of the s-GDH mutations described above. Thus, the present invention also includes an isolated polynucleotide sequence encoding the s-GDH mutant protein described above.

The invention further comprises an expression vector comprising the nucleic acid sequence according to the invention operably linked to a promoter sequence capable of directing its expression in a host cell.

The invention further comprises an expression vector comprising a nucleic acid sequence according to the invention operably linked to a promoter sequence capable of directing its expression in a host cell. Preferred vectors are plasmids such as pACSGDH shown in FIGS. 2 and 3.

Expression vectors useful in the present invention typically include all or part of the origin of replication, antibiotic resistance for selection, promoter for expression and s-GDH gene variants. Expression vectors may also contain other DNA sequences known in the art, such as signal sequences (for better folding, transport or secretion into the plasma membrane space), inducers for better modulation of expression, or segments for cloning. Site may be included.

The nature of the expression vector chosen should be affinity with the host cell to be used. For example, when cloning in an E. coli cell system, the expression vector should include a promoter (eg, lac or trp ) isolated from the genome of E. coli. Suitable origins of replication, such as ColE1 plasmid origin of replication, can be used. Suitable promoters include, for example, lac and trp . It is also preferred that the expression vector comprises a sequence encoding a selection marker, such as an antibiotic resistance gene. As the selection marker, ampicillin resistance, or kanamycin resistance may be conveniently used. All such materials are known in the art and are commercially available.

Appropriate expression vectors comprising the desired coding and regulatory sequences can be constructed using standard recombinant DAN techniques known in the art, many of which are described in Sambrook et al, in "Molecular Cloning: A Laboratory Manual" (1989). Cold Spring Harbor, NY, Cold Spring Harbor Laboratory Press.

The present invention further relates to a host cell comprising an expression vector comprising a DNA sequence encoding part or all of the mutant s-GDH. The host cell preferably comprises an expression vector comprising some or all of one of the DNA sequences with one or more mutations shown in Examples 2-8. Suitable host cells are described, for example, in E. coli HB 101 (ATCC 33694), Stratagene (11011 North Torrey Pine Road, La Jolla, CA, USA), commercially available from Pomega (2800 Woods Hollow Road, Madison, WI, USA). XLl-Blue MRF 'commercially available from, etc. are included.

Expression vectors can be introduced into host cells by a variety of methods known in the art. For example, transformation of a host cell with an expression vector can be performed by a polyethylene glycol mediated protoplast transformation method (Sambrook et al. 1989, supra). However, other methods of introducing expression vectors into host cells, such as electroporation, biolistic injection or protoplasm fusion, can also be used.

After the expression vector comprising the s-GDH variant is introduced into an appropriate host cell, the host cell can be cultured under conditions that allow expression of the desired s-GDH variant. Host cells comprising the desired expression vector having a DNA sequence encoding part or all of the s-GDH mutation can be readily identified by antibiotic selection. Expression of s-GDH variants can be confirmed by different methods such as measuring production of s-GDH mRNA transcription, immunological detection of gene products or detection of enzymatic activity of gene products. Preferably enzymatic analysis is applied.

The present invention also teaches the generation and screening of s-GDH variants. Random mutagenesis and saturation mutagenesis are performed as known in the art. Variants are screened for substrate specificity for glucose (activity to glucose compared to maltose) and KM values. Selected assay conditions are adapted so that the expected small improvement, for example caused by a single amino acid substitution, can be measured. One method of selecting or screening appropriate mutations is provided in Example 3. In this way any change or improvement compared to the wild type enzyme can be clearly detected.

Of course, it should be understood that not all expression vectors and DNA regulatory sequences work equally well in expressing the DNA sequences of the present invention. In addition, not all host cells work equally well with the same expression system. However, those skilled in the art will make appropriate choices among expression vectors, DNA regulatory sequences, and host cells with the instructions provided herein without undue experimentation.

The invention also relates to a method of preparing the s-GDH variant of the invention comprising culturing the host cell of the invention under conditions suitable for the production of the s-GDH mutation of the invention. For bacterial host cells, typical culture conditions are liquid media comprising a carbon and nitrogen source, appropriate antibiotics and inducers (depending on the expression vector used). Typical suitable antibiotics include ampicillin, kanamycin, chloroamphenicol, tetracycline and the like. Typical inducers include IPTG, glucose, lactose and the like.

It is preferred that the polypeptide of the invention is prepared and obtained in a host cell expressing a DNA sequence encoding a mutant s-GDH. Polypeptides of the invention can also be obtained by in vitro translation of mRNA encoded by a DNA sequence encoding a mutant s-GDH. For example, the DNA sequence can be synthesized as described above and inserted into an appropriate expression vector, which can then be used in an in vitro transcription / translation system.

Expression vectors comprising isolated polynucleotides as defined and described above, operably linked to a promoter sequence capable of promoting expression in a cell free peptide synthesis system represent another preferred embodiment of the present invention.

For example, polypeptides produced by the methods described above can then be isolated and purified using several routine protein purification techniques. For example, chromatographic methods such as ion exchange chromatography, gel filtration chromatography, and affinity chromatography can be used.

One of the major applications of the improved s-GDH variants of the present invention is for use in test strips that monitor blood-glucose levels in diabetic patients. Insensitivity of PQQ-dependent glucose dehydrogenase to oxygen has a great advantage over glucose oxidase, as described above. More importantly, because the s-GDH variant has improved specificity for glucose and significantly reduced enzymatic activity for other sugars, interference with maltose, galactose and / or other sugars that may be present in the sample to be analyzed is eliminated. Significantly reduced. Of course, different kinds of samples can be irradiated. Body fluids such as serum, plasma, serous fluid or urine are preferred sources for such samples.

The invention also includes a method for detecting, determining or measuring glucose in a sample using the s-GDH variant mutations according to the invention. It is particularly preferred that the improved method for detecting glucose in a sample is characterized in that the glucose detection, determination or measurement is carried out using a sensor or test strip device.

Also within the scope of the present invention are devices for detecting or measuring glucose in a sample comprising the s-GDH mutation according to the present invention, and other reagents required for said measurement.

S-GDH with improved substrate specificity of the present invention is also a biosensor for on-line monitoring of glucose in a reactor or sample (D'Costa, EJ, et al., Biosensors 2 (1986) 71-87; Laurinavicius, V. , et al., Analytical Letters 32 (1999) 299-316; Laurinavicius, V., et al., Monatshefte fuer Chemie 130 (1999) 1269-1281; Malinauskas, A. et al., Sensors and Actuators, B: Chemical 100 (2004) 395-402). For this purpose, the s-GDH variant is an oxygen-insensitive glassy electrode (e.g., Ye et al., 1993), for more accurate measurement of glucose concentration, for example, with an osmium complex comprising a redox conductive epoxy network. Can be used for coating).

In addition, there are other possible applications of s-GDH variants with improved substrate specificity according to the present invention. For example, such s-GDH variants may be used in the production of aldonic acid. Wild type s-GDH has a high conversion in substrate oxidation to produce gluconic acid and other aldonic acids. By using s-GDH variants that are more specific for glucose, the preparation of gluconic acid will result in even fewer byproducts. With other s-GDH variants with different substrate specificities, it is possible to produce different aldonic acids as needed.

In the examples below, all reagents, restriction enzymes, and other materials were obtained from Roche Diagnostics Germany, unless otherwise indicated by commercial sources, and used according to the instructions provided by the supplier. Operations and methods used for purification, characterization and cloning of DNA are well known in the art (Ausubel, F., et al, in "Current protocols in molecular biology" (1994) Wiley Verlag), Can be retrofitted accordingly.

The following examples further illustrate the invention. These examples are not intended to limit the scope of the present invention, but to further understand the present invention.

Example  One

E. In Coli  Wild type A. Calcoaceticus  Availability PQQ  Dependent Glucose  D Hydrogenase Cloning  And expression

The s-GDH gene was isolated from the Acinetovector chalcocetiticus strain LMD 79.41 according to standard methods. The wild type s-GDH gene was subcloned into a plasmid containing a mgl promoter for controllable expression (see patent application WO 88/09373). The new construct was called pACSGDH (see FIGS. 2 and 3). Recombinant plasmids were introduced into host organisms selected from the E. coli group. These organisms were then cultured under appropriate conditions and colonies showing s-GDH activity were selected.

Plasmid pACSGDH was isolated according to the manufacturer's protocol from 200 ml overnight cultures of the above mentioned clones using the QIAGEN Plasmid Maxi Kit (Qiagen). The plasmid was resuspended in 1 ml of distilled water. Plasmid concentrations were measured using a Beckman DU 7400 Photometer.

The yield was 600 μg. Plasmid quality was determined by agarose gel electrophoresis.

Example  2

T348G  Mutation Generation

Mutant s-GDH with T348G mutation was prepared as an important starting template for the generation of insertional variants. The s-GDH mutation was chosen due to the reduced activity of maltose compared to glucose (see WO 02/34919).

The threonine at position 348 was replaced with glycine using The QuickChange Site-Directed Mutagenesis Kit (Stratagene, Cat. 200518). Appropriate primers were designed.

The 5'- and 3'- primers used for mutagenesis are complementary to each other and included a modified codon at the central position for exchange of threonine to glycine (ACA to AGG). 12-16 nucleotides were located on each side of these nucleotides. The sequence of nucleotides was identical to the sense and anti-sense DNA-strands flanking the codons for amino acid exchange. Instead of ACA = threonine for sense and TGT codons for anti-sense strand, primers included CCC for GGG = glycine and anti-sense strand for sense (see SEQ ID NOs: 3 and 4).

PCR-response and DpnI digestion were performed according to the instructions. Thereafter, 1 μl of sample was used for electroporation of XL-MRF′-cells. Electroporation was achieved at 2.5 KV in a 0.2 cm cuvette using BioRad E. Coli Pulser (BioRad). After 1 hour of growth at 37 ° C. in 1 ml LB, bacteria were plated in LB-ampicillin agar plates (100 μg / ml ampicillin) and grown at 37 ° C. overnight. Mutant s-GDH clones were examined using the following screening method.

Example  3

Screening

Mutant colonies on the agar plates described above were imprinted into microtiter plates (MTP) containing 200 μl LB-Ampicillin-medium / well and incubated overnight at 37 ° C. This plate is called master plate.

From each master plate, transfer 5 μl samples per well to MTP containing 5 μl B (B = Bacterial Protein Extraction Reagent; Pierce No. 78248) per well for cell disruption, and for the activity of s-GDH, 240 μl of 0.0556 mM pyrrolo-quinoline quinone (PQQ); 50 mM Hepes; 15 mM CaCl ₂ pH 7.0 / well was incubated at 25 ° C. for 2 hours and at 10 ° C. overnight. The plate is called a working plate.

3 × 10 μl samples per hole from the working plate were transferred to three empty MTPs. One was then tested with glucose at standard concentration, the second with a reduced glucose concentration (1.9 mM instead of 30 mM), and the third with maltose or other selected sugar molecules as substrate. All other sugar molecules selected were used at equimolar standard concentrations, ie 30 mM. For all assays, 90 μl of mediator solution (see Example 7) already containing the sugar to be analyzed was applied.

dE / min was calculated and the value using 30 mM glucose as substrate was set to 100% activity. Values obtained with other sugars were compared to glucose values and calculated as percent activity (eg, for maltose: dE / min maltose / dE glucose) * 100). This is equivalent to the crossreactivity of the (variant) enzyme.

The value obtained with 1.9 mM glucose was compared with the 30 mM glucose value and calculated as percent activity (dE / min 1.9 mM glucose / 30 mM glucose) * 100. This gives the% -value, which is an indirect measure of KM value for the variant being analyzed. According to this calculation, higher% -values indicate lower (= better) KM values.

The following mutations have been identified:

enzyme M / G (per 30 mM,%) Active 1,9 / 30 mM glucose,% Amino acid exchange WT 105 70% - Mutation 25-30% 21% T348G

Example  4

Mutations s- from Designated Site Mutations GDH  Gene sequencing

 Isolate plasmids containing genes for the s-GDH T348G mutation with 25-30% maltose / glucose cross-reactivity (High Pure Plasmid Isolation Kit, Roche Diagnostics GmbH, No. 1754785), ABI Prism Dye Terminator Sequencing Kit and ABI Sequencing was performed using 3/73 and 3/77 sequencing (Amersham Pharmacia Biotech).

The following primers were used:

Sense strand: GDH 1: 5'-TTA ACG TGC TGA ACA GCC GG-3 '(= SEQ ID NO: 5)

GDH 2: 5'-ATA TGG GTA AAG TAC TAC GC -3 '(= SEQ ID NO: 6)

result:

The desired mutations on DNA and amino acid levels were achieved, resulting in a change from T to G at position 348 (mature protein). No further mutations on the gene were found.

Example  5

T348G  Mutation based insertion Variant  produce

Insert variants were generated using the QuickChange Site-Directed Mutagenesis Kit (Stratagene, Cat. 200518). Appropriate primers were designed for insertion between amino acids at positions 428 and 429 (see SEQ ID NO: 2) and PCR was performed with mutations in s-GDH (T348G mutation, see Example 4) according to the manufacturer's instructions.

For the primers, each codon at the insertion site was randomly synthesized to obtain all 20 possible amino acid exchanges. These codon nucleotides flanked 11 to 13 nucleotides at each end.

Sense strand:

in429X_F: 5'- CTGCCGGAAATNNNGTCCAAAAAGATG -3 '

(= SEQ ID NO: 7)

Anti-sense strands:

in429X_R 5'- CATCTTTTTGGACNNNATTTCCGGCAG -3 '

(= SEQ ID NO: 8)

PCR reactions and DpnI digestion were performed according to the instructions. 1 μl of each reaction was then used for electroporation of XL1F-cells as described above. After 1 hour of growth in 1 ml LB at 37 ° C., bacteria were plated in LB-ampicillin agar plates (100 μg / ml ampicillin) and grown overnight at 37 ° C. The screening method described in mutant s-GDH clones was performed. To make sure statistically that variants with 20 possible amino acid insertions were screened, 200 clones were tested. Plasmid isolation was performed on clones with altered substrate specificity and sequencing as described above.

result:

Table 1:

Example  6

To maltose  Compared to glucose on  Generation of additional insertion mutations with high substrate specificity for

In WO 02/34919 several amino acid exchanges at different positions of s-GDH have been found to improve substrate specificity for glucose, for example compared to maltose. Combination of amino acid exchange T348G with amino acid substitutions at other positions, such as positions 169, 171, 245, 341, and / or 349, further improved substrate specificity. The two mutations described above, with substrate specificities for maltose / glucose of 3,5 and 7%, respectively, were selected to effect insertion according to the invention (proline in this case) between positions 428 and 429. Insertion was accomplished using the primers of SEQ ID NOs: 9 and 10.

SEQ ID NO: 9 insP429_F:

5'-GATACTGCCGGAAATCCAGTCCAAAAAG -3 '

SEQ ID NO: 10 insP429_R:

5'-CTTTTTGGACTGGTCCTCCGGCAGTATC -3 '

Plasmid DNA isolation of the template, site mutagenesis PCR, electroporation, screening, DNA sequencing of selection mutations were performed as described above.

result:

It is clear from the table that the maltose / glucose substrate specificity has changed significantly. Maltose conversion was found to decrease from 105% to 2-4% compared to maltose / glucose conversion of wild type s-GDH. C / 1 and D / 1 mutations were examined in detail.

Example  7

Wild type or variant s- GDH  Analysis of Purification and Enzyme Activity for Each

Grown cells (LB-Amp. 37 ° C.) were harvested and suspended in potassium phosphate buffer pH 7.0. Cell disruption was performed by French Press passage (700-900 bar). After centrifugation, the supernatant was applied to an S-Sepharose (AmershamBiosciences) column equilibrated with 10 mM potassium phosphate buffer pH 7.0. After washing, s-GDH was eluted using a 0-1 M NaCl salt gradient. Fractions showing s-GDH activity were collected, separated for potassium phosphate buffer pH 7.0 and chromatographed again on a re-equilibrated S-Sepharose column. The active fractions were pooled and gel filtration was performed using a Superdex ^® 200 column (Amersham Biosciences). The active fractions were collected and stored at -20 ° C.

Purified wild type and variant s- GDH  Enzyme analysis and protein measurement for each:

Protein measurement was performed by Pierce, Protein Assay Reagent no. It was performed using 23225 (calibration curve with BSA, 30 minutes, 37 ° C.).

0.0556 mM pyrrolo-quinoline quinone (PQQ); 50 mM Hepes; Samples were diluted to 1 mg protein / ml with 15 mM CaCl ₂ pH 7.0 and incubated at 25 ° C. for 30 minutes for reconstitution or activity.

After incubation, the samples were transferred to 50 mM Hepes; 15 mM CaCl ₂ Dilute to pH 7.0, up to about 0,02 U / ml, and 50 μl of each diluted sample was added to 0.315 mg (4- (dimethylphosphinylmethyl) -2-methyl-pyrazolo- [1.5a]-as a medium. To 1000 μl of 0.2 M citrate buffer (pH 5.8; 25 ° C.) containing imidazol-3-yl)-(4-nitrosophenyl) -amine (see patent US 5,484,708) / ml and 30 mM sugar.

Absorbance at 620 nm was monitored at 25 ° C. for the first 5 minutes.

One unit enzyme activity corresponds to a conversion of 1 mMol mediator / min under the assay conditions.

Calculation: activity = (total volume * dE / min [U / ml]): (ε * sample volume * 1)

(ε = coefficient of absorbance; ε _{620 nm} = 30 [l * mmol ⁻¹ * cm ⁻¹ ]).

The analysis was performed with glucose and maltose (Merck, Germany) respectively.

result:

It is evident from the table that the novel variants C / 1 and D / 1, including the insertion between positions 428 and 429 of s-GDH, show further improvement on maltose / glucose cross-reactivity. Despite the several amino acid substitutions and insertions, the enzyme activity on glucose per mg / protein still exceeds 10% of the corresponding wild type enzyme activity.

Example  8

Maltose  Glucose in the presence or absence of  Measure

The C / 1 and D / 1 variants of wild type s-GDH and s-GDH can be applied for glucose measurement in the presence or absence of maltose, respectively. Reference sample contains 50 mg glucose / dl. The "test" sample contains 50 mg glucose / dl and 100 or 200 mg / dl maltose, respectively. The same amount of GDH (U / ml; see enzyme assay above) activity is used for each assay.

 Mix the following in the cuvette:

1 ml 0.315 mg (4- (dimethylphosphinylmethyl) -2-methyl-pyrazolo- [1.5a] -imidazol-3-yl)-(4-nitrosophenyl) -amine ml / 0.2 M citrate pH 5.8

0.033 ml reference or test sample

The assay was initiated by adding 0.050 ml s-GDH (excess s-GDH to glucose conversion) to the cuvette. The change in absorption at 620 nm was monitored. After 2-5 minutes, constant values were observed and dE / 5 minutes were calculated. The value obtained by measuring the reference sample with wild type s-GDH was set to 100%. Other values were calculated in% compared to the above reference values.

result:

Significantly less maltose interference was detected in the test samples when using the novel variants. It is clear that the concentration of s-GDH to best distinguish glucose from maltose in the sample can be further optimized. Too much enzyme increases maltose conversion and too little enzyme will not reach the end point of absorption as it does not convert all glucose.

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                         SEQUENCE LISTING <110> Roche Diagnostics GmbH        F. Hoffmann-La Roche AG   <120> Genetically engineered pyrroloquinoline quinone dependent glucose         dehydrogenase comprising an amino acid insertion <130> 22669 WO <150> EP 04017069 <151> 2004-07-20 <160> 11 <170> PatentIn version 3.2 <210> 1 <211> 1362 <212> DNA <213> Acinetobacter calcoaceticus <220> <221> CDS (222) (1) .. (1362) <400> 1 gat gtt cct cta act cca tct caa ttt gct aaa gcg aaa tca gag aac 48 Asp Val Pro Leu Thr Pro Ser Gln Phe Ala Lys Ala Lys Ser Glu Asn 1 5 10 15 ttt gac aag aaa gtt att cta tct aat cta aat aag ccg cac gcg ttg 96 Phe Asp Lys Lys Val Ile Leu Ser Asn Leu Asn Lys Pro His Ala Leu             20 25 30 tta tgg gga cca gat aat caa att tgg tta act gag cga gca aca ggt 144 Leu Trp Gly Pro Asp Asn Gln Ile Trp Leu Thr Glu Arg Ala Thr Gly         35 40 45 aag att cta aga gtt aat cca gag tcg ggt agt gta aaa aca gtt ttt 192 Lys Ile Leu Arg Val Asn Pro Glu Ser Gly Ser Val Lys Thr Val Phe     50 55 60 cag gta cca gag att gtc aat gat gct gat ggg cag aat ggt tta tta 240 Gln Val Pro Glu Ile Val Asn Asp Ala Asp Gly Gln Asn Gly Leu Leu 65 70 75 80 ggt ttt gcc ttc cat cct gat ttt aaa aat aat cct tat atc tat att 288 Gly Phe Ala Phe His Pro Asp Phe Lys Asn Asn Pro Tyr Ile Tyr Ile                 85 90 95 tca ggt aca ttt aaa aat ccg aaa tct aca gat aaa gaa tta ccg aac 336 Ser Gly Thr Phe Lys Asn Pro Lys Ser Thr Asp Lys Glu Leu Pro Asn             100 105 110 caa acg att att cgt cgt tat acc tat aat aaa tca aca gat acg ctc 384 Gln Thr Ile Ile Arg Arg Tyr Thr Tyr Asn Lys Ser Thr Asp Thr Leu         115 120 125 gag aag cca gtc gat tta tta gca gga tta cct tca tca aaa gac cat 432 Glu Lys Pro Val Asp Leu Leu Ala Gly Leu Pro Ser Ser Lys Asp His     130 135 140 cag tca ggt cgt ctt gtc att ggg cca gat caa aag att tat tat acg 480 Gln Ser Gly Arg Leu Val Ile Gly Pro Asp Gln Lys Ile Tyr Tyr Thr 145 150 155 160 att ggt gac caa ggg cgt aac cag ctt gct tat ttg ttc ttg cca aat 528 Ile Gly Asp Gln Gly Arg Asn Gln Leu Ala Tyr Leu Phe Leu Pro Asn                 165 170 175 caa gca caa cat acg cca act caa caa gaa ctg aat ggt aaa gac tat 576 Gln Ala Gln His Thr Pro Thr Gln Gln Glu Leu Asn Gly Lys Asp Tyr             180 185 190 cac acc tat atg ggt aaa gta cta cgc tta aat ctt gat gga agt att 624 His Thr Tyr Met Gly Lys Val Leu Arg Leu Asn Leu Asp Gly Ser Ile         195 200 205 cca aag gat aat cca agt ttt aac ggg gtg gtt agc cat att tat aca 672 Pro Lys Asp Asn Pro Ser Phe Asn Gly Val Val Ser His Ile Tyr Thr     210 215 220 ctt gga cat cgt aat ccg cag ggc tta gca ttc act cca aat ggt aaa 720 Leu Gly His Arg Asn Pro Gln Gly Leu Ala Phe Thr Pro Asn Gly Lys 225 230 235 240 tta ttg cag tct gaa caa ggc cca aac tct gac gat gaa att aac ctc 768 Leu Leu Gln Ser Glu Gln Gly Pro Asn Ser Asp Asp Glu Ile Asn Leu                 245 250 255 att gtc aaa ggt ggc aat tat ggt tgg ccg aat gta gca ggt tat aaa 816 Ile Val Lys Gly Gly Asn Tyr Gly Trp Pro Asn Val Ala Gly Tyr Lys             260 265 270 gat gat agt ggc tat gct tat gca aat tat tca gca gca gcc aat aag 864 Asp Asp Ser Gly Tyr Ala Tyr Ala Asn Tyr Ser Ala Ala Ala Asn Lys         275 280 285 tca att aag gat tta gct caa aat gga gta aaa gta gcc gca ggg gtc 912 Ser Ile Lys Asp Leu Ala Gln Asn Gly Val Lys Val Ala Ala Gly Val     290 295 300 cct gtg acg aaa gaa tct gaa tgg act ggt aaa aac ttt gtc cca cca 960 Pro Val Thr Lys Glu Ser Glu Trp Thr Gly Lys Asn Phe Val Pro Pro 305 310 315 320 tta aaa act tta tat acc gtt caa gat acc tac aac tat aac gat cca 1008 Leu Lys Thr Leu Tyr Thr Val Gln Asp Thr Tyr Asn Tyr Asn Asp Pro                 325 330 335 act tgt gga gag atg acc tac att tgc tgg cca aca gtt gca ccg tca 1056 Thr Cys Gly Glu Met Thr Tyr Ile Cys Trp Pro Thr Val Ala Pro Ser             340 345 350 tct gcc tat gtc tat aag ggc ggt aaa aaa gca att act ggt tgg gaa 1104 Ser Ala Tyr Val Tyr Lys Gly Gly Lys Lys Ala Ile Thr Gly Trp Glu         355 360 365 aat aca tta ttg gtt cca tct tta aaa cgt ggt gtc att ttc cgt att 1152 Asn Thr Leu Leu Val Pro Ser Leu Lys Arg Gly Val Ile Phe Arg Ile     370 375 380 aag tta gat cca act tat agc act act tat gat gac gct gta ccg atg 1200 Lys Leu Asp Pro Thr Tyr Ser Thr Thr Tyr Asp Asp Ala Val Pro Met 385 390 395 400 ttt aag agc aac aac cgt tat cgt gat gtg att gca agt cca gat ggg 1248 Phe Lys Ser Asn Asn Arg Tyr Arg Asp Val Ile Ala Ser Pro Asp Gly                 405 410 415 aat gtc tta tat gta tta act gat act gcc gga aat gtc caa aaa gat 1296 Asn Val Leu Tyr Val Leu Thr Asp Thr Ala Gly Asn Val Gln Lys Asp             420 425 430 gat ggc tca gta aca aat aca tta gaa aac cca gga tct ctc att aag 1344 Asp Gly Ser Val Thr Asn Thr Leu Glu Asn Pro Gly Ser Leu Ile Lys         435 440 445 ttc acc tat aag gct aag 1362 Phe Thr Tyr Lys Ala Lys     450 <210> 2 <211> 454 <212> PRT <213> Acinetobacter calcoaceticus <400> 2 Asp Val Pro Leu Thr Pro Ser Gln Phe Ala Lys Ala Lys Ser Glu Asn 1 5 10 15 Phe Asp Lys Lys Val Ile Leu Ser Asn Leu Asn Lys Pro His Ala Leu             20 25 30 Leu Trp Gly Pro Asp Asn Gln Ile Trp Leu Thr Glu Arg Ala Thr Gly         35 40 45 Lys Ile Leu Arg Val Asn Pro Glu Ser Gly Ser Val Lys Thr Val Phe     50 55 60 Gln Val Pro Glu Ile Val Asn Asp Ala Asp Gly Gln Asn Gly Leu Leu 65 70 75 80 Gly Phe Ala Phe His Pro Asp Phe Lys Asn Asn Pro Tyr Ile Tyr Ile                 85 90 95 Ser Gly Thr Phe Lys Asn Pro Lys Ser Thr Asp Lys Glu Leu Pro Asn             100 105 110 Gln Thr Ile Ile Arg Arg Tyr Thr Tyr Asn Lys Ser Thr Asp Thr Leu         115 120 125 Glu Lys Pro Val Asp Leu Leu Ala Gly Leu Pro Ser Ser Lys Asp His     130 135 140 Gln Ser Gly Arg Leu Val Ile Gly Pro Asp Gln Lys Ile Tyr Tyr Thr 145 150 155 160 Ile Gly Asp Gln Gly Arg Asn Gln Leu Ala Tyr Leu Phe Leu Pro Asn                 165 170 175 Gln Ala Gln His Thr Pro Thr Gln Gln Glu Leu Asn Gly Lys Asp Tyr             180 185 190 His Thr Tyr Met Gly Lys Val Leu Arg Leu Asn Leu Asp Gly Ser Ile         195 200 205 Pro Lys Asp Asn Pro Ser Phe Asn Gly Val Val Ser His Ile Tyr Thr     210 215 220 Leu Gly His Arg Asn Pro Gln Gly Leu Ala Phe Thr Pro Asn Gly Lys 225 230 235 240 Leu Leu Gln Ser Glu Gln Gly Pro Asn Ser Asp Asp Glu Ile Asn Leu                 245 250 255 Ile Val Lys Gly Gly Asn Tyr Gly Trp Pro Asn Val Ala Gly Tyr Lys             260 265 270 Asp Asp Ser Gly Tyr Ala Tyr Ala Asn Tyr Ser Ala Ala Ala Asn Lys         275 280 285 Ser Ile Lys Asp Leu Ala Gln Asn Gly Val Lys Val Ala Ala Gly Val     290 295 300 Pro Val Thr Lys Glu Ser Glu Trp Thr Gly Lys Asn Phe Val Pro Pro 305 310 315 320 Leu Lys Thr Leu Tyr Thr Val Gln Asp Thr Tyr Asn Tyr Asn Asp Pro                 325 330 335 Thr Cys Gly Glu Met Thr Tyr Ile Cys Trp Pro Thr Val Ala Pro Ser             340 345 350 Ser Ala Tyr Val Tyr Lys Gly Gly Lys Lys Ala Ile Thr Gly Trp Glu         355 360 365 Asn Thr Leu Leu Val Pro Ser Leu Lys Arg Gly Val Ile Phe Arg Ile     370 375 380 Lys Leu Asp Pro Thr Tyr Ser Thr Thr Tyr Asp Asp Ala Val Pro Met 385 390 395 400 Phe Lys Ser Asn Asn Arg Tyr Arg Asp Val Ile Ala Ser Pro Asp Gly                 405 410 415 Asn Val Leu Tyr Val Leu Thr Asp Thr Ala Gly Asn Val Gln Lys Asp             420 425 430 Asp Gly Ser Val Thr Asn Thr Leu Glu Asn Pro Gly Ser Leu Ile Lys         435 440 445 Phe Thr Tyr Lys Ala Lys     450 <210> 3 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Primer sequence for T348G sense strand <400> 3 catttgctgg ccaggggttg caccgtcat 29 <210> 4 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Primer sequence for T348G antisense strand <400> 4 atgacggtgc aacccctggc cagcaaatg 29 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Sequencing primer GDH 1 <400> 5 ttaacgtgct gaacagccgg 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Sequencing primer GDH 2 <400> 6 atatgggtaa agtactacgc 20 <210> 7 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Wobbled insertion primer in429X_F sense strand <220> <221> misc_feature (222) (12) .. (14) N is a, c, g, or t <400> 7 ctgccggaaa tnnngtccaa aaagatg 27 <210> 8 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Wobbled insertion primer in429X_R anti sense strand <220> <221> misc_feature (222) (14) .. (16) N is a, c, g, or t <400> 8 catctttttg gacnnnattt ccggcag 27 <210> 9 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> insP429 <400> 9 gatactgccg gaaatccagt ccaaaaag 28 <210> 10 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> insP429_R <400> 10 ctttttggac tggtcctccg gcagtatc 28 <210> 11 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> synthetic peptide <220> <221> misc_feature (222) (3) .. (3) <223> Xaa can be any naturally occurring amino acid <400> 11 Gly Asn Xaa Val Gln Lys Asp 1 5  

Claims (22)

A soluble form of EC 1.1.99.17, also known as PQQ-dependent soluble glucose dehydrogenase (s-GDH), wherein the variant is known from A. calcoaceticus (SEQ ID NO: 2) substitution of one amino acid residue selected from alanine, glycine or serine to threonine at position 348 between insertions of one amino acid residue selected from phenylalanine, leucine, methionine or proline between positions 428 and 429 of the s-GDH wild type sequence Variants further comprising. delete The variant of claim 1, wherein said inserted amino acid is proline. delete delete The variant of PQQ-dependent s-GDH according to claim 1, wherein asparagine at position 428 is substituted with one amino acid residue selected from leucine, proline or valine. delete delete The variant of claim 1, further comprising a substitution of one amino acid selected from alanine, methionine or glycine for tyrosine at position 171. The variant of claim 1, further comprising substitution of glutamic acid at position 245 with one amino acid selected from aspartic acid, asparagine or glutamine. The variant of claim 1, further comprising a substitution of one amino acid selected from valine, alanine, leucine or isoleucine at methionine at position 341. The variant of claim 1, further comprising a substitution of one amino acid selected from phenylalanine, tyrosine, or tryptophan with leucine at position 169. The variant of claim 1, further comprising a substitution of alanine or glycine for valine at position 349. An isolated polynucleotide encoding the s-GDH variant protein according to claim 1. An expression vector comprising said isolated polynucleotide as defined in claim 14 operably linked to a promoter sequence capable of promoting expression of the polynucleotide in a host cell. A host cell comprising the expression vector of claim 15. A method of preparing an s-GDH variant, the method comprising culturing the host cell of claim 16 under conditions suitable for preparing an enzyme variant. An expression vector comprising an isolated polynucleotide as defined in claim 14 operably linked to a promoter sequence capable of promoting expression in a cell free peptide synthesis system. delete A method of detecting, determining or measuring glucose in a sample using the s-GDH variant according to claim 1, wherein said improvement comprises contacting the sample with said variant. 21. The method of claim 20, wherein said detection, determination, or measurement of glucose is further performed using a sensor or test strip device. Device for the detection or measurement of glucose in a sample comprising the s-GDH variant according to claim 1.

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